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ABSTRACT. The genetic diversity and genetic relationship of the two main groups of African sheep, thin-tailed and fat-tailed sheep, represented by the indigenous Tunisian sheep breeds "Barbarine" (BAR, fat-tailed) and "Queue Fine de l'Ouest" (QFO, thin-tailed) were investigated. The genotypes of 110 animals belonging to these two breeds and their crossbreed (CRO) were assessed using 17 microsatellite markers. The results showed high levels of genetic diversity and a total of 256 alleles were identified in the whole population. The mean values of observed and expected heterozygosity were 0.719 and 0.789, respectively, and the mean allelic richness estimate was 10.89. values over all loci indicated a notable level of inbreeding within the whole population. However, the F ST value (0.007) showed a low level of genetic differentiation between these two native breeds. The high level of both gene flow and molecular coancestry coefficient detected between the two breeds and their CRO revealed an old miscegenation between the BAR and QFO breeds. The clustering analysis performed with the STRUCTURE software confirmed gene flow between these two breeds. Results arising from this study provide evidence regarding the genetic structure and variability of the two main local sheep breeds, and the implications of their actual management, which indicates the need for an urgent conservation strategy in order to prevent significant gene flow and preserve the remaining breed specificity for future generations.